Functional analysis of candidate genes in primary T cell immunodeficiencies. Tracing the molecular etiologies of patients with severe combined immunodeficiency (SCID) or other T lymphopenic diseases (variant SCID and combined immunodeficiency, CID) has led to the identification of many novel genes that play critical roles in normal development. With the advent of Next Gen Sequencing (NGS), identification of the causative mutations in SCID is becoming more readily achievable; however, in some cases NGS identifies numerous candidate mutations that must be screened to identify the causative mutation. Distinguishing among these candidates in such cases is too labor-intensive to accomplish using mouse models. Consequently, we propose to identify such mutations using a rapid screening process in zebrafish. As we and others have shown, hematopoiesis in zebrafish is highly conserved, such that developmental abnormalities caused by the elimination of essential zebrafish genes can be complemented by their mammalian orthologs. Accordingly, we propose to screen novel candidate SCID genes by: 1) knocking down their expression in zebrafish to determine if this blocks T cell development; and 2) determining if re- expression of the wild type, but not mutant, mammalian ortholog, restores development. We have recently demonstrated that this can be done rapidly and effectively (Zhang et al., 2013). After identification of a likely disease gene by this means, we will employ zinc-finger nuclease mutagenesis to create knockout mouse models lacking the identified genes and knockin models replicating the patients' mutation(s). This project will be pursued as a joint effort with Dr. Puck (UCSF), who has a repository of T lymphopenic patients for whom numerous candidate disease genes have been identified. These patients were identified through the California SCID newborn screening program, for which she serves as Immunology Consultant. Treatment of SCID is far less effective if initiated after the disease becomes manifest due to infections. However, because more than 80% of SCID cases have no prior family history, diagnosing pre-symptomatic SCID patients is particularly challenging. To circumvent this problem, Dr. Puck and others have developed an effective population-based, screening test to identify T lymphopenia. This test entails the quantitation of T cell receptor excision circles (TRECs) in DNA extracted from the dried blood spots already used for universal newborn screening. After 32 months of screening 1.3 million infants in California, (with an incidence of T cell lymphopenia about 1/20,000 births), Dr. Puck has identified 10 T lymphopenic patients without a known SCID genotype for whom numerous candidate genes have been identified by NGS. We propose to screen the function of candidate genes from these 10 patients as well as additional patients identified during the 24 month course of this grant.